Protein-tyrosine sulfation is mediated by one of only two Golgi tyrosylprotein sulfotransferases in the human and mouse genome (TPST-1 and TPST-2) that sulfate an unknown number of secreted and transmembrane proteins that transit the secretory pathway. Tyrosine sulfation plays a role in protein-protein interactions in several well-defined systems. However, only several dozen tyrosine-sulfated proteins have been described and for many, the functional importance of sulfotyrosine residues remains unknown. Many more tyrosine-sulfated proteins are likely to exist and await description. The pace of discovery of tyrosine-sulfated proteins has been very slow. There have been three major barriers to developing a full understanding of the importance of tyrosine sulfation in biological systems. First, there has been the lack of knockout animal models to study the biology of this enzyme system. Second, there has been the lack of a facile means to both identify additional tyrosine-sulfated proteins and probes to explore the role of tyrosine sulfation in protein function. Finally, a robust analytical method to determine the site of tyrosine sulfation in proteins has not been available. We developed Tpst1 and Tpst2 knockout mice and found that Tpst2-/- males are infertile, whereas Tpst1-/- males have normal fertility. The male infertility phenotype is characterized by normal spermatogenesis, but abnormal sperm function. These findings suggest that one or more proteins expressed in the male genital tract must be tyrosine-sulfated for normal sperm function. However, no proteins directly or indirectly involved in sperm function are known to be tyrosine- sulfated. We have developed a novel anti-sulfotyrosine monoclonal antibody that recognizes sulfotyrosine residues in proteins independent of the sequence context and which allows the detection and purification of tyrosine-sulfated proteins from complex biological samples. In addition, we have developed a novel subtractive mass spectrometry based method to determine the sites of tyrosine sulfation in proteins that is broadly applicable. We will exploit these novel tools and methods to identify tyrosine-sulfated proteins using the male genital tract as a model system in order to fully validate the approach, to refine our methodology, and to explore the role of tyrosine sulfation in male reproductive biology. Three specific aims are proposed. Aim 1 - Identify tyrosine-sulfated proteins expressed in the male genital tract. Expression profiles of tyrosine-sulfated proteins in the male genital tract of wild type, Tpst1-/-, and Tpst2-/- mice will be determined by PSG2 Western blotting in combination with subcellular fractionation. Tyrosine-sulfated proteins will then be purified from wild type mice by PSG2 affinity chromatography and identified by microsequencing. Aim 2 The presence of sulfotyrosine and/or the site(s) of sulfation in the proteins identified in Aim 1 will be determined using our subtractive mass spectrometry strategy and other independent methodology. Aim 3 Identify the protein(s) that are under sulfated in Tpst2-/- mice and the enzymatic basis for defective sulfation of these proteins. PUBLIC HEALTH RELEVANCE: We propose to apply novel tools and methodology to identify tyrosine-sulfated proteins in the male genital tract. Our studies will catalyze studies in many areas of investigation by expanding the known TPST substrate repertoire, providing novel insights into structure-function relationships of proteins expressed in the male genital tract, and providing insights into the molecular basis for male infertility in Tpst2-/- mice. Our methods we will be broadly applicable to other system and/or model organism and facilitate a rapid expansion in our understanding of the role of tyrosine sulfation in biologic systems and human diseases. [unreadable] [unreadable] [unreadable]